Project Summary The gastrointestinal (GI) tract is a dynamic ecosystem, regulating nutrient absorption, infection, commensalism, and immunity in vibrant homeostasis. The GI tract includes physical, chemical, molecular, and immune barrier to infection. Despite these barriers, enteroviruses, including coxsackievirus B3 (CVB3), successfully transit the GI barrier to initiate infection, spread throughout the host causing disease in diverse tissues, and finally be shed in stool. The essential molecular and anatomic features of the GI tract that restrict or render hosts susceptible to enterovirus infection are only beginning to be understood. Additionally, host barriers dramatically affect pathogen population dynamics. This is fundamentally important because intrahost enterovirus population dynamics directly affect pathogenesis. However, the effect of the GI tract on CVB3 population dynamics is completely unexplored. The long-term objectives for this project is to define the intestinal molecular and cellular pressures that effect CVB3 evolution and pathogenesis. The immediate goals of this project are to: 1) precisely map the kinetics, tissues, and cells involved in CVB3 replication in the GI tract; 2) assess CVB3 population dynamics, including founding population size, in the intestine; 3) test the role of host factors, including interferon and sex, on intestinal CVB3 replication and population dynamics. These will be accomplished in the following specific aims: Specific Aim 1: Categorize the cell types CVB3 infects in the intestine of male and female mice by A) identifying specific infected cell types by cell sorting, B) determining infection site and kinetics using reporter viruses, C) assessing the role of immune cells on intestinal infection by depleting specific cells. Specific Aim 2: Examine CVB3 population dynamics and routes of dissemination in male and female mice by A) developing and validating a library of isogenic barcoded CVB3 strains, B) creating a spatio-temporal atlas of CVB3 population dynamics by sequencing viruses from tissues and specific cell types, and C) depleting immune cells and assessing spread and transmission of CVB3. The knowledge gained in this work will be a critical step to understanding the role of the GI tract in modulating CVB3 genetics, evolution, and pathogenesis.